Determination of L-glutathione by spot test and spectrophotometric methods based on its interaction with phenazine

This research paper presents simple and quick eco-friendly spot test and spectrophotometric methods for the determination of L-glutathione. The spot test assay is based on the formation of a color complex with phenazine methosulphate and L-glutathione on a thin-layer chromatography plate followed by image analysis using a scanner as a detector. For analysis, the image was converted into red, green, and blue (RGB) histograms. A series of parameters that influenced the color formation were investigated, and under the optimal conditions, a good linearity was observed in the range of 200-1000 μg mL^-1 and 249-1000 μg mL^-1 of L-glutathione with correlation coefficients of 0.9907 for B and 0.9903 for G channels. For the spectrophotometric method, a good linearity was obtained in the range of 2.1-60 μg mL^-1 of L-glutathione concentration with a correlation coefficient of 0.9961. A mechanism of the reaction of L-glutathione with phenazine was proposed and confirmed by Fourier transform infrared and mass spectroscopy.